Radiometals offer distinct advantages over iodine when used to label monoclonal antibodies. Radiometals that bind to antibodies by chelation help avoid the deleterious effects of oxidation experienced in common iodination reactions. Labeling with metals also overcomes the problems of in-vivo deiodination by tumor and normal tissues, particularly when using rapidly internalized antibodies. Radiometals also provide a wealth of choices of half-life and emissions for various applications (diagnosis or therapy).
Radiometals can generally be attached to antibodies by the use of a "bifunctional chelate" which is first covalently attached to the antibody to form an antibody-chelate conjugate which then binds the radiometal. The most commonly used chelate is DTPA and it is generally conjugated to antibodies via bicyclic DTPA anhydride, which forms a covalent amide bond between an antibody amine and one of the carboxylic acid groups of DTPA [Hnatowich, et al., Science, 220, 613 (1983)]. This method, however, has drawbacks. The use of this procedure yields high liver retention and slow body clearance [Goodwin, J. Nucl. Med., 28, 1358 (1987)], as well as a substantial amount of crosslinked antibody (two antibodies linked together by a DTPA bridge). This crosslinking increases liver retention and decreases tumor uptake.
The use of liposome-encapsulated .sup.111 In-.sup.14 C-DTPA showed that the .sup.14 C-DTPA is cleared through the kidney into the urine while the .sup.111 In remains in the liver [Mathias, et al. J. Nucl. Med., 28, 657 (1987)]. This suggests that the retention of indium is due to its transchelation in the liver following its detachment from DTPA, indicating that chelates which form stronger complexes with .sup.111 In will be necessary in order for the .sup.111 In-chelate complex to survive liver catabolism of the antibody-chelate conjugate and be excreted intact through the kidney into the urine. The introduction of benzyl groups onto the backbone of EDTA and DTPA has been shown to increase the serum stability of .sup.111 In-EDTA and -DTPA chelates and antibody conjugates [see, for example, Cole, et al., Nucl. Med. Biol., 13, 363 (1986)].
In spite of these improvements, benzyl EDTA and benzyl DTPA are not the optimal chelates for all radiometals. For example, copper-67 1-(p-isothiocyanatobenzyl)EDTA labeled antibodies are very unstable in serum and require the use of macrocyclic ligands [Cole, et al., J. Nucl. Med., 28, 83 (1987)] or porphyrins [Mercer-Smith, et al., Los Alamos National Laboratory Report LA-10709-PR, 58 (1986)] to form complexes that are stable in serum.
As discussed above, one strategy followed to overcome the problems caused by liver catabolism is to use chelates that form more stable radiometal complexes on the immunoconjugate so that the radiometal chelate can survive the liver metabolism of the antibody intact. A second approach to this problem is to place a metabolizable linking group between the antibody and radiometal chelate so that the linker is rapidly cleaved, thereby reducing the time the chelate resides in the liver and the opportunity for transchelation with hepatic proteins. Several groups have been investigating this approach. Faster whole body clearance and higher tumor/tissue ratios have been obtained with linkers containing a disulfide or an ester between the antibody and DTPA-p-(aminoethyl) anilide [Paik, et al., J. Nucl. Med., 28, 572 (1987)]. Linking groups containing either a disulfide, ester, thioether, thiorea, or peptide group have been placed between the antibody and the para-amino group of p-aminobenzyl EDTA. While the disulfide clearance is promising it would appear that the disulfide clearance occurs in the circulation and may be too rapid for use with antibodies.
One object of the present invention is the preparation of ligands that will form radiometal chelates that are capable of surviving in-vivo. This is accomplished by combining the rigidity of the ligand with the general utility of polyaminocarboxylates such as EDTA and DTPA, for example by the substitution of a rigid molecule such as trans-1,2-diaminocyclohexane for an ethylene diamine portion of a polyaminocarboxylate. The rigidity of the cyclohexane ring fixes two of the nitrogens into a position where they can readily complex radiometals. The semi-rigid chelate cyclohexyl EDTA is known and has been used in the preparation of copper complexes [Robinson, Prog. Nucl. Med., 4, 80 (1978)]. The present invention relates to new semi-rigid chelates that bind the radiometal, can be conjugated to monoclonal antibodies, and overcome the stability problems of the prior art materials.